It had been long believed that enzyme was protein until the discovery of ribozyme, an RNA segment with endoribonuclease activity, in 1981. The discovery has changed the conventional belief dramatically. Thomas Cech found that the precursor of a ribosomal RNA (rRNA), inTetrahymera (protozoan) undergoes self-splicing in the absence of proteins. The intron (IVS) in this precursor RNA molecule is precisely removed by a catalytic activity of the RNA itself (Nature, 308: 820-825, 1984). Subsequently, catalytically active RNA molecules that act for the cleavage of phosphodiester bond have been found one after another. Symons has found conserved ribozyme sequence in several plant viruses and Haseloff and Gerlach have constructed a synthetic ribozyme with a catalytic domain of 24 bases (Nature, 334: 585-591, 1988).
FIG. 1 shows the structure of a synthetic ribozyme comprising a binding segment C which recognizes the sequence of an RNA molecule (substrate) and forms a base-pairing with the substrate, and a catalytically active segment B containing a specific 24-nucleotide [SEQ ID No: 1]. The site A contains sequences in the substrate, immediately adjacent to the site of cleavage (indicated by arrow). The site A can be other bases, although FIG. 1 shows GUC.
Haseloff, J., et al., constructed synthetic ribozymes as follows: The ribozyme sequences were synthesized as single-stranded oligonucleotides. The oligonucleotides were ligated to plasmid DNA, and after transformation, DNA of the resulting clones were digested with restriction enzyme. The plasmids containing a sequence encoding synthetic ribozymes were recovered. The plasmids were used after linearization for transcription in vitro. Then, synthetic enzymes were obtained. They designed three types of oligomers encoding three different ribozymes and demonstrated that these ribozymes cleaved the CAT gene transcript at three different sites.
The conventional method, called a run-off method, requires an extra step (restriction enzyme digestion) as described below. The recombinant plasmid containing the DNA fragment encoding a gene of interest is first digested with restriction enzyme at the 3' end of the gene and then the fragment is used for in vitro transcription to give RNA enzyme. When the recombinant plasmid is transcribed without restriction enzyme digestion, an extra flanking sequence is also attached at 3' side of the transcribed gene product. The digestion step is therefore designed for removing the extra sequence at 3' side. In order to avoid the extra segment at 5' side the DNA fragment at 5' end is designed to be located immediately downstream of a promoter sequence. For the DNA fragment at 3' end, there has been no alternative method but a run-off method using restriction enzyme since no universal terminator has been found (FIG. 2). Haseloff, J., et al., also synthesized ribozymes using the run-off method.
Hereafter, the gene of interest to be transcribed is a trans-acting ribozyme. However, the discussion is general and applicable to any sequences &lt;genes). Although a ribozyme has been obtained through restriction enzyme digestion and subsequent in vitro transcription using a linearized DNA as a template (run-off transcription), this method cannot be used in vivo since there is no restriction enzyme which specifically cuts at 3' end of the ribozyme-coding DNA template. In addition, an extra nucleotide sequence is found bound to the sequence of the ribozyme after transcription unless the 5' end of the DNA fragment encoding ribozyme is placed immediately downstream of a promotor. The conventional method therefore has three major points to improve. Firstly, a digestion step in the procedure can be eliminated. Secondly, the ribozyme can be amplified in vivo while the host cell harboring the recombinant DNA encoding the ribozyme is kept growing. Thirdly, the extra nucleotide sequence can be removed from the ribozyme.
An object of the invention provides a recombinant DNA containing novel synthetic ribozyme sequences which can be utilized as a template being an intact closed circular form without restriction enzyme digestion. The recombinant DNA can be produced in vivo while the host cell is kept growing and produces a ribozyme free of an extra nucleotide sequence.
Moreover, when ribozymes are to be used as anti-HIV agents (Sarver, M., Cantin E., Chang, P., Ladne, P., Stephens, D., Zaia, J. & Rossi, J. (1990) Science 247, 1222-1225), one has to consider genetic variability of HIV. HIV is infamous for its high mutation rate caused by the low fidelity of its reverse transcriptase which lacks the function of proof-reading and has a tendency to add an extra nucleotide upon DNA template jumps. This mutability of HIV not only makes it difficult to prepare vaccines against HIV but also hinders the application of ribozymes toward HIV RNA cleavage because of the high substrate-specificity of the ribozyme. One way to overcome this mutability of HIV in applying ribozymes is to target several conserved sites simultaneously. Then, even if one or more sites undergo mutations to avoid cleavages by ribozymes, the other conserved sites can potentially be cleaved by other ribozymes which have been targeted to those sites.
There seems to be at least two ways to express multitargeted-ribozymes: the simpler way is just to join several sequences of ribozymes possessing different target sites in tandem so that all the transcribed multitargeted-ribozymes would be connected in tandem into a single RNA (connected-type); the other strategy is to combine cis-acting ribozymes with trans-acting ribozymes so that the several trans-acting ribozymes targeted to HIV (or any other sequences) would be trimmed at both 5' and 3'-ends by the actions of cis-acting ribozymes, liberating several trans-acting ribozymes which would work independently of others (shotgun-type). However, kinetic behaviors of the above mentioned two types of expression systems for multitargeted-ribozymes are unknown and need to be examined.